Effects of methyl jasmonate elicitation on the carvone and limonene contents, phenolic compounds and antioxidant activity in caraway (Carum carvi L.) callus cultures

Abstract This study investigated the elicitation effects of several methyl jasmonate (MeJ) concentrations (0, 25, 50, and 100 µM) on various biochemical traits of caraway (Carum carvi L.) callus cultures. The 25 μM MeJ concentration yielded the highest callus growth rate (0.57 mm day−1), total flavonols content (2.58 mg QE g−1 FW) and total carotenoids content (0.04 μg g−1 FW), whereas the highest relative fresh weight (75.72%), total phenolics content (76.90 mg GAE g−1 FW), total flavonoids content (58.49 mg QE g−1 FW) and phenylalanine ammonia lyase activity (3.40 nmol cinnamic acid mg−1 h−1 FW) were obtained with the 50 μM MeJ concentration. The highest antioxidant activity through DPPH assay (8.37%) and malondialdehyde content (7.82 μmol g−1 FW) were observed at 100 μM MeJ. The HPLC conducted 21 days post-elicitation revealed a 5.55-fold increase of carvone accumulation (1.83 μg g−1 DW) at 50 μM MeJ and a 2.7-fold increase (0.62 μg g−1 DW) of limonene at 50 μM MeJ. The optimal dosages applied for carvone and limonene accumulation under MeJ elicitation could be used to scale up the economic production of these elite medicinal compounds through caraway callus cultures. Graphical Abstract


Introduction
Caraway (Carum carvi L.) is a biennial herb and belongs to the Apiaceae family (Hammami et al. 2018).It is an important medicinal plant often used as a spice around the globe (Teuscher et al. 2006;Bitterling et al. 2021).According to traditional medicine, it has the potential to be an antiseptic, antidiabetic, carminative, stimulant and antibacterial herb (Synowiec et al. 2019;Bouyahya et al. 2021).Carvone (50-60%) and R-(þ)-limonene (35-45%) make up more than 90% of the entire essential oil content of caraway, and are used as precursors for the synthesis of other flavorings and fine chemicals (Bitterling et al. 2021).
Secondary metabolites (SMs) (e.g.phenolics, flavonoids and anthocyanins) play an important antioxidant role in plants (Yang et al. 2018), and are of special interest to the medical industry as they participate in defense mechanisms and scavenge reactive oxygen species (ROS) (Yang et al. 2018).
Methyl jasmonate (MeJ) is a methyl ester known for regulating plant growth and response to environmental tensions (Nabi et al. 2021).Very few studies have shown the effects of in vitro elicitation on certain important pharmaceutical compounds, such as carvone (Narayani and Srivastava 2017;Dehghani-Aghchekohal et al. 2022) pcymene, thymol andcarvacrol (Dehghani-Aghchekohal et al. 2022) Considering the low yield of carvone and limonene from the seed essential oil, the in vitro production of these compounds seems to be a suitable substitutive method.
This study (a) researched the optimization procedure for callus induction of Carum carvi L. with high efficiency, (b) examined the effects of MeJ elicitation on the various biochemical traits of Carum carvi L. calli for the first time in literature, and (c) established an optimized concentration of MeJ that enhanced the major bioactive compounds (carvone and limonene) under callus elicitation.

Results and discussion
Different treatments showed different percentages of germination, ranging from 13.33% to 93% (Table S1).The MS media supplemented with NAA (2 mg L À1 ) þ BAP (0.5 mg L À1 ) was recognized as the best combination for highest callus induction (94.8%) and CGR (0.43 mm day À1 ) in the hypocotyl explants (Table S2).The highest CGR (0.57 mm day À1 ) was observed at 25 mM MeJ concentration, which was nonsignificant when compared with the control (Table 1).The relative fresh weight (RFW) showed significant variation, from 75.72 mm day À1 at 50 mM MeJ to 23.44 mm day À1 at 100 mM MeJ (Table 1).After the elicitation process, the color of the calli changed to light brown with greenish spots (Figure S1).A significant increase of malondialdehyde (MDA) was observed, from 2.63 lmol g À1 FW in the control to 7.82 lmol g À1 FW in calli treated with 100 mM MeJ (Table 1).The highest (76.9 mg GAE g À1 FW) and lowest (48.40 mg GAE g À1 FW) total phenolics content (TPC) values were observed in the 50 mM MeJ and control treatments, respectively (Table 1).When compared with the control, the TPC increased 1.37-, 1.58-and 1.10-fold in callus cultures treated with the 25, 50, and 100 mM MeJ concentrations, respectively (Table 1).The total flavonoids (TFD) content varied from 33.16 mg QE g À1 FW in control, up to 58.49 mg QE g À1 FW in the 50 mM MeJ treatment (Table 1).The total flavonols (TFL) content showed variation from 1.10 mg QE g À1 FW in the control, up to 2.58 mg QE g À1 FW in the 25 mM MeJ treatment (Table 1).The anthocyanins value varied from 125.40 mmol g À1 FW under the non-elicitation condition to 625.90 mmol g À1 FW under the 50 mM MeJ concentration (Table 1), revealing a 5-fold increase compared with the control (Table 1).The carotenoids content significantly increased under 25 mM MeJ (0.041 lg g À1 FW) when compared with the control (0.024 lg g À1 FW) (Table 1).A direct dose-response relationship was observed between MeJ and the antioxidant activity of the caraway calli (Table 1).The activity of PAL increased from 1.45 nmol CA mg À1 h À1 FW in the control to 3.40 nmol CA mg À1 h À1 FW in the 50 mM MeJ treatment, where its mean value showed a 6-fold increase (Figure S2).Carvone accumulation increased significantly in the 25 and 50 mM MeJ-elicited callus cultures (Figure S3a).When compared with the control, carvone accumulation in calli treated with 50 mM MeJ increased approximately 5.55-fold (Figure S3a).The limonene contents of the 25, 50 and 100 mM MeJ elicitations increased 2-, 2.7-and 2.34-fold, respectively, compared with the control (Figure S3b).
Callus elicitation processes have been used to produce SMs for various industrial purposes in short periods of time and independent of environmental conditions (Namdeo 2007).Here, the callus induction of caraway was first optimized as a preliminary step to develop callus cultures for further experiments.This study demonstrated that the combined effects of both cytokinins and auxins [NAA (2 mg L À1 ) þ BAP (0.5 mg L À1 )], with pre-dominant concentrations of auxins, promoted a high percentage of callus initiation from the hypocotyl explants of caraway.Methyl jasmonate impacted on the morphological traits of the calli (color and texture) by increasing the browning and softening of the callus tissue.Furthermore, the beneficial effects of MeJ could be seen with the increase of cell division (e.g. increase in callus growth rate) and water absorption (e.g. increase in relative fresh weight).The effects of MeJ elicitation on the growth and fresh weight of the calli revealed a dose dependent trend.The stressor effects of MeJ (especially at concentrations higher than 50 mM) on RFW is probably due to the inhibitory effects of higher MeJ concentrations (> 50 mM) on cell growth and capacity of cell osmotic adjustment (Yazdanian et al. 2022).The generation of ROS under environmental stress causes the peroxidation of membrane lipids, which leads to membrane damage and subsequently, the production of MDA (Das and Roychoudhury 2014).Similar results were reported in studies on Panax ginseng (Ali et al. 2007) and A. jesdanium (Yazdanian et al. 2022).The positive significant correlation between MDA and DPPH (0.92 ÃÃ ) (Figure S4) demonstrated a significant increase of antioxidants produced in the calli to scavenge ROS under MeJ induced conditions.An increase in phenolics accumulation was observed as a defense mechanism of caraway in response to MeJ elicitation.The significant increase of phenolics content may be due to the triggering effects of MeJ on the signal transduction pathways for phenolics synthesis (Biondi et al. 2004;Samadi et al. 2019).Our findings confirm the positive effects of MeJ on the increase of the TFL content in A. jesdianum (Yazdanian et al. 2022), and TFD content increase in the cell suspension cultures of yew (Jalalpour et al. 2014), Panax ginseng (Ali et al. 2007) and Thevetia peruviana (Mendoza et al. 2018).This significant increase of TFD could be specifically due to the probable positive effects of the 50 mM MeJ concentration on the enhanced expression of genes that contribute to the synthesis pathway of the TFD.The novel results of this study are consistent with the findings of other researchers who investigated the dose-dependence of anthocyanins on various MeJ concentrations in cell cultures, such as Ram et al. (2013) on Rosa hybrid and Suan See et al. (2011) on Melastoma malabathricum.According to our findings, the carotenoid accumulation responses depended on a lower MeJ concentration (25 mM).It was observed that under MeJ elicitation, the antioxidant activity in caraway was not dose-dependent.This phenomenon could be due to the sufficient effects of the different MeJ levels on the stimulation of callus antioxidant activity against ROS.Interestingly, the positive correlation between DPPH activity and TFD (0.64 Ã ) demonstrated high antioxidant activity of flavonoid compounds in the calli of these species upon elicitation.Phenylalanine ammonia-lyase (PAL) plays an important role in the activation of defense mechanisms and the biosynthesis of many phenolic compounds (MacDonald and D'Cunha 2007).In this regard, the significant correlation between TPC and PAL (0.74 ÃÃ ), and TFD and PAL (0.70 Ã ) (Figure S4), showed that the main phenolic compounds with the phenolic structure in caraway were accumulated via an activity increase of these involving key enzymes in the biosynthesis of phenolic compounds at the cellular level.An increase in PAL activity was observed in the caraway calli in response to MeJ elicitation.Similarly, the increasing effect of MeJ on PAL activity was seen in the callus cultures of stevia (Samadi et al. 2019), and the cell suspension cultures of Hypericum perforatom (Wang et al. 2015), Salvia miltiorrhiza (Zhao et al. 2010), and Taxus spp.(Wang and Wu 2005).Taking into account the economic value of carvone and limonene, it should be stated that adequate extraction of these compounds requires field cultivation for a long growth period, and subsequently, a laborious process for its extraction.Our findings confirm the acceptable yield of these two compounds through callus culture elicitation.The positive significant correlation between limonene and DPPH (0.84 ÃÃ ) (Figure S4) suggested that the antioxidant activity of limonene in the caraway calli was higher compared with carvone, and that this specific monoterpenoid effectively contributed to antioxidant activity at the cellular level.It seems that a one-by-one analysis might be required to get an exact understanding of the modulating effects of MeJ on the production of carvone and limonene at the cellular level.

Experimental section
Please see the supplementary material.

Conclusion
The elicited callus cultures of caraway at the 50 mM MeJ concentration offered an effective method to significantly improve the production of carvone and limonene in short periods of time for their potential use in the pharmaceutical and industrial fields.
For future research, it is suggested that studies be conducted on the regulation of enzymes involved in the biosynthesis of limonene and carvone at the callus level.The present study could be efficiently utilized to enhance the mass production of carvone and limonene from C. carvi L. on commercial scales.

Table 1 .
Mean comparison of different studied traits of caraway under callus elicitation by methyl jasmonate.